Alexander J. Stevenson

Self-Renewal and High Proliferative Colony Forming Capacity of Late-Outgrowth Endothelial Progenitors is Regulated by Cyclin-Dependent Kinase Inhibitors Driven by Notch Signaling

Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34− ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self‐renew in stringent single‐cell cultures and gave rise to both CD34 + and CD34− cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin‐dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ‐secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self‐renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self‐renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self‐renewal potential. Stem Cells 2016;34:902–912

Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer

Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7–expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers. Mol Cancer Ther; 14(12); 2753–61. ©2015 AACR.

Decatenation checkpoint-defective melanomas are dependent on PI3K for survival.

Melanoma cell lines are commonly defective for the G2‐phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint‐defective cell lines were three‐fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF‐05212384 promoted apoptosis in the checkpoint‐defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint.

Inhibition of Aurora A and Aurora B Is Required for the Sensitivity of HPV-Driven Cervical Cancers to Aurora Kinase Inhibitors

The activity and efficacy of Aurora inhibitors have been reported in a wide range of cancer types. The most prominent Aurora inhibitor is alisertib, an investigational Aurora inhibitor that has been the subject of more than 30 clinical trials. Alisertib has inhibitory activity against both Aurora A and B, although it is considered to be primarily an Aurora A inhibitor in vivo. Here, we show that alisertib inhibits both Aurora A and B in vivo in preclinical models of HPV-driven cervical cancer, and that it is the inhibition of Aurora A and B that provides the selectivity and efficacy of this drug in vivo in this disease setting. We also present formal evidence that alisertib requires progression through mitosis for its efficacy, and that it is unlikely to combine with drugs that promote a G2 DNA damage checkpoint response. This work demonstrates that inhibition of Aurora A and B is required for effective control of HPV-driven cancers by Aurora kinase inhibitors. Mol Cancer Ther; 16(9); 1934–41. ©2017 AACR.

Genome-Wide Overexpression Screen Identifies Genes Able to Bypass p16-Mediated Senescence in Melanoma

Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene (HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.

Mechanism of action of the third generation benzopyrans and evaluation of their broad anti-cancer activity in vitro and in vivo

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.

Endogenous replication stress marks melanomas sensitive to CHK1 inhibitors in vivo

Purpose: Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity in vitro and in vivo. Here, we have investigated the molecular basis of this activity. Experimental Design: We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 in vitro and in vivo. The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines. Results: A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death in vitro, and the drug effectively inhibits tumor growth in vivo. In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i. Conclusions: CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress in vivo. Clin Cancer Res; 24(12); 2901–12. ©2018 AACR.

Abstract A33: Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo

CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress. Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as single agents in melanoma and lung cancer. We have found that sensitivity in vitro and in vivo to single agent CHK1 inhibitor is loss of the S phase cell cycle checkpoint response. This is through a number of mechanisms including the uncoupling of CHK1 activation with the destabilization of CDC25A. Loss of checkpoint by over-expressing components of the checkpoint or inhibition of Wee1, covert CHK1 inhibitor insensitive cells to sensitive, and similarly depletion of CDC25A reduces CHK1 inhibitor sensitivity in sensitive lines. Loss of the S phase checkpoint provides cells with an adaptive advantage through introduction of moderate levels of genomic instability. The increased DNA damage found with CHK1 inhibitor treatment is not sufficient to induce cell death, but also involves a mechanism that is dependent in part on DNA-PK activity. Loss of S phase checkpoint function is predicted for Note: This abstract was not presented at the conference. Citation Format: Zay Yar Oo, Alexander Stevenson, Catherine Lanagan, Loredana Spoerri, Jill Larsen, Brian Gabrielli. Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A33.

Abstract 5102: TRXE-009-1 has pan-acting anticancer activity and potently inhibits colony formation of prostate cancer epithelial cell explants

Background: TRXE-009-1 is a novel small molecule from the super benzopyran (SBP) drug family under development as an intravenous cytotoxic agent. Cantrixil (TRXE-002-1), our lead SBP candidate targeting ovarian cancer, recently achieved Investigational New Drug (IND) status and is undergoing safety assessment in a multi-center Phase I clinical trial. Our aim was to investigate pathways associated with response to TRXE-009-1 and identify cancer types for clinical translation. Methods: Eurofins Oncopanel240 with multiplex analysis and bioinformatic assessment was used to identify cytotoxic and pathway response across a panel of cancer types. Western blot and live cell imaging were used to investigate protein levels and cell cycle progression. Results: TRXE-009-1 demonstrated broad cytotoxic activity across a large panel of cell lines and was particularly active against kidney (RCC), liver (HCC), head and neck cancer, lung cancer, sarcoma, prostate cancer, osteosarcoma, and neuroblastoma. Further, TRXE-009-1 combined synergistically with two targeted agents used extensively in the clinical treatment of RCC patients. Cleaved caspase 3/7 and G2/M arrest were strongly associated with TRXE-009-1 activity. Live imaging confirmed G2/M arrest and implicated cytoskeleton disruption in TRXE-009-1 induced cell death. When compared to 43 reference oncology compounds, TRXE-009-1 clustered with a known epigenetic modifier. Additional analysis indicated that, compared to the least sensitive lines, the most sensitive cancer cell lines were more likely to be positive for LIN28B, a stemness marker associated with aggressive cancers. This is in line with historical studies suggesting that SBPs may preferentially target cancer-stem like cells. Using a panel of prostate cancer epithelial explants in a colony forming assay, we confirmed strong activity against colony-initiating cells. Studies are current to confirm protein changes associated with SBP activity and to further investigate the impact of TRXE-009-1 on the cytoskeleton. In vivo studies previously established activity of TRXE-009-1 against melanoma, prostate, and brain cancer and additional studies are current to optimize the delivery formulation to be used in preclinical safety studies prior to clinical progression. Conclusion: Given that cancer stem-like cells are typically more chemoresistant and are thought to support tumor recurrence, the activity of TRXE-009-1 against this subpopulation of cancer cells makes it a particularly promising candidate for further development. Citation Format: Eleanor I. Ager, Dominika Butler, Alex Stevenson, Eiffe Eiffe, Andrew Heaton, Brian Gabrielli, Norman J. Maitland, David Brown. TRXE-009-1 has pan-acting anticancer activity and potently inhibits colony formation of prostate cancer epithelial cell explants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5102. doi:10.1158/1538-7445.AM2017-5102

Abstract 945: synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA HPV has been identified as the definitive agent in cancers of the cervix, penis, vulva, vagina, anus, skin, eye, and head and neck, and is responsible for 5% of the total cancer burden worldwide. HPV oncogenes disable a number of tumour suppressor pathways, including p53 and Rb, contributing to the transformed phenotype. We have performed an siRNA screen using the kinome (779 genes) library to identify genes that when depleted are synthetically lethal with HPV transformation. The primary and validations screens have confirmed Aurora A kinase (AURKA) as a potential synthetic lethal target selective for HPV transformed cells. In vitro research using the investigational selective small molecule AURKA inhibitor alisertib found alisertib to be significantly more potent towards the HPV transformed cells, and selectively promoted apoptosis in the HPV cancers. The drug was shown to target the HPVE7 oncogene, the level of expression of this oncogene possibly influencing sensitivity. Apoptosis was sensitive to Mcl-1 but not Bcl-2 over expression, indicating that the mechanism is associated with the proteolytic destruction of Mcl-1 in the extended mitosis in the alisertib treated HPV cancer lines. Xenograft experiments with cervical cancer cell lines showed alisertib inhibited growth of HPV and non-HPV xenografts during treatment. The non-HPV cancer growth was delayed, but in two separate HPV cancers models, regression and no resumption of growth was detected at even 50 days post-treatment. A second transgenic model of premalignant disease driven solely by HPVE7 similarly demonstrated sensitivity to drug treatment. These findings provide preclinical evidnce that alisertib warrants evaluation as a potential targeted compound with activity in HPV-transformed cervical cancer and premalignant disease that may have application to other HPV driven cancers. Citation Format: Brian G. Gabrielli, Fawzi Bokhari, Max Ranall, Zay Yar Oo, Alex Stevenson, Weili Wang, Sara McKee, Graham Leggatt, Paul Leo, Thomas J. Gonda, Nigel AJ McMillan. Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 945. doi:10.1158/1538-7445.AM2015-945